The share of biopharmaceuticals has been rapidly increasing in a medicinal market. Among biopharmaceuticals, remarkably increasing have been recombinant protein formulations such as enzymes, hormones, antibodies, growth factors, and blood coagulation factors. For stable supplies of these pharmaceuticals, establishment of a system for producing a recombinant protein, which is safe, low-cost and efficient, is desired.
Recombinant proteins have been conventionally expressed using Escherichia coli, etc. because of the productivity and efficiency. However, the expression systems of E. coli have problems that it is difficult to reproduce the conformation of a protein and that a post-translational modification such as glycosylation modification cannot be achieved. Thus, many of those recombinant protein formulations each comprising a cytokine, an enzyme, an antibody drug, or the like, which involves its conformation or the post-translational modification for its activity, are produced using Chinese Hamster Ovary (CHO) cells.
The expression systems using CHO cells also involve problems. Until recent years, a serum or a biological material derived from a heterologous animal has been used for culturing CHO cells. However, the use of the serum or the biological material derived from the heterologous animal causes problems about safety such as a risk of infection of a virus originated from an animal and an allergy due to a heterologous animal antigen. Further, a problem concerning stability, such as a lot-to-lot variability, is also caused by using biological materials. Therefore, chemically defined media (synthetic media) have been developed, in which media components produced by chemical syntheses or recombinant techniques are used instead of biological materials such as a serum (Non-patent Literature 1: Sunstrom, et al., 2000). However, those components produced by chemical syntheses or recombinant techniques, especially growth factors, are expensive and unstable. Thus, it is desirable for industrial production to conduct culture without adding such substances or growth factors.
An adapted culture method, in which cells are gradually adapted to an environment which is free of materials derived from biologics or growth factors, is an approach to eliminate those factors from CHO cell culture. It has been reported for a long time that cells have an ability to adapt to an environment and can be adapted to an environment with only minimum essential nutrients by spending time on adaptation (Non-patent Literature 2: Kagawa, et al., 1969; Non-patent Literature 3: Kagawa, et al., 1970). In a protein expression system, when a protein of interest is expressed by transfecting the CHO cells with a vector carrying cDNA of the protein, a drug resistant gene is used in the transfection in order to select cells carrying the gene of interest. If any transfection is carried out in addition to transfection for introducing the gene for the protein of interest, a selection method comes to be limited. Thus, it is desirable not to carry out additional transfection. In the adapted culture method, it is unnecessary to perform an additional manipulation for gene modification such as gene introduction, because cells themselves are being adapted to the environment. In this meaning, the adapted culture method has a high flexibility in terms of introduction of the gene of interest. However, the adapted culture method is time-consuming and labor-intensive, and has a low success rate. Thus, this method has not been tried in order to obtain adapted cells derived from the CHO cells having high productivity.
Further, the CHO cells also involve other problems. The CHO cells are inherently adherent cells and therefore they are not suitable for tank culture by using, e.g., a bioreactor, which is used in large-scale productions of materials for industrial use. Adherent cells require a large cell-adhering surface area because they propagate while adhering to a vessel wall. To ensure such large adhering area, a high-density culture apparatus of a layered or hollow fiber type, or an adherent carrier such as a micro-carrier, is used, which causes problems such as complication of the culture apparatus and increase in production costs. Furthermore, to suspend the cells, a carrier or a flotation agent such as a surfactant may be used. Such carrier increases the costs for production. On the other hand, surfactants are cytotoxic and often exert toxicity to cells. Further, those surfactants must be removed as impurities upon purification of the product, and may also inhibit the purification. Therefore, it has been desired to suspend the CHO cells without using such flotation agents.